Effect Of MiR-491 On EMT Inhibiting Invasion And Metastasis Of HCC

Objective:The recombinant adenoviral vector overexpressing miR-491 was constructed and transfected into HCCLM6 cell line.The HCCLM6 cells were subcultured to detect the expression of miR-491 in tumor cells,and the proliferation and adhesion of hepatoma cells were observed.And changes in invasive ability,Western blot and immunofluorescence were used to measure the changes of E-cadherin,N-cadherin,Vimentin,MMP2,GIT1,ERK levels,and to evaluate the effect of miR-491 expression on the proliferation,adhesion and invasion of hepatoma cells.The effects of key molecules related to EMT-related protein levels and their pathways,and the effect of over-expression of miR-491 on the invasion and metastasis of HCC by EMT.Methods:(1)Construction of an adenoviral vector overexpressing miR-491 designing and synthesizing an ADV2-shRNA DNA template,and annealing the template,constructing a vector,and sequencing the same to obtain the correct sequence,and then using adenovirus packaging.(2)Continuous expansion and cryopreservation of HCCLM6 cells(3)HCCLM6 cell transfection:cell passage amplification,cell plating,according to different MOI values,the corresponding virus solution amount is calculated according to the virus titer and mixed with the medium containing the final concentration of 5 ug/ml of polybrene;After 48 hours,the survival of each group of cells was observed and the expression of mir-491 in each group was detected(4):Culture and passage of HCCLM6 cells:The frozen HCCLM6 cells were quickly thawed,added to the medium,centrifuged,and the supernatant was discarded.The cells were then transferred to a culture flask and placed in a constant temperature incubator.to cultivate.The growth of the cells was observed by microscope.(5):Detection of cell proliferation,adhesion and invasion ability:The proliferation,adhesion and invasion ability of each group of cells were detected by CCK8 method,adhesion test and Transwell chamber method.(6):qRT-PCR detection:Real-time quantitative reverse transcription PCR(qRT-PCR)was used to detect the expression levels of GIT1 and ERK in each treatment group.(7)Western-blot test:collect cell samples,select over-expression group,empty vector group and blank control group,extract total cell protein,and perform EMT after electrophoresis separation,transformation,development and gray value calculation.Changes in levels of related proteins and channel proteins,and comparison of differences between groups.(8)Immunofluorescence detection:EMT-related proteins and invasion-related molecules are detected in cell samples by immunofluorescence staining techniques.Results:(1)By successfully constructing the miR-491 overexpression vector and transfecting it into the hepatoma cell line HCCLM6,the qRT-PCR method was used to detect the miR-491 expression change between the groups,and it was found that there was a significant difference between the groups.The expression of the staining group(overexpression group)was significantly higher than that of the empty vector group and the blank control group,and the difference was statistically significant.(2)Through the subculture of HCCLM6 liver cancer cells,a large number of liver cancer cells were collected,and it was observed that the cell morphology of the overexpressing group was significantly attenuated from the epithelial morphology to the spindle-shaped mesenchymal morphology(3)The CCK8 method,adhesion test and Transwell chamber method were used to measure the proliferation,adhesion and invasion ability of each group of cells.It was found that there were significant differences,and the difference was statistically significant.Overexpression group and blank control group and empty vector group In comparison,its proliferation and invasion ability are significantly reduced,and the adhesion ability is enhanced.(4)qRT-PCR method showed significant differences in the expression changes between GIT1 and ERK groups.The expression levels of GIT1 and ERK in the overexpression group were significantly decreased.And the difference is statistically significant.(5)Western-blot detection showed that the epithelial marker protein E-cadherin was higher in the overexpression group than in the blank control group and the empty vector group,while the levels of the interstitial marker proteins N-cadherin and Vimentin were lower than those in the blank control group and the empty vector group.The expression of MMP2 in the invasive marker protein was lower than that in the blank control group and the empty vector group.The channel protein GIT1 and ERK were decreased compared with the blank control group and the empty vector group,and the difference was statistically significant.(6)Immunofluorescence indicated that the expression of each protein was different.The expression of Vimentin and N-cadherin in the overexpressed;group was significantly decreased,the E-cadherin was increased,and the MMP2 was significantly decreased.Conclusions:(1)High expression of miR-491 effectively inhibits the proliferation and invasion of HCCLM6 hepatoma cells and enhances adhesion.(2)The mechanism of high expression of miR-491 inhibiting invasion and metastasis of HCC is related to the key proteins E-cadherin,N-cadherin,Vimentin,which EMT(3)miR-491 may regulate EMT process through GIT 1/ERK and affect MMP2 inhibition of liver cancer invasion.